Research ArticleImproved Healing of Diabetic Foot Ulcer upon OxygenationTherapeutics through Oxygen-Loading NanoperfluorocarbonTriggered by Radial Extracorporeal Shock Wave

Shunhao Wang,1,2 Chunyang Yin,1,2 Xiaoguang Han,3 Anyi Guo,3 Xiaodong Chen,4

Sijin Liu ,1,2 and Yajun Liu 3

1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences, Beijing 100085, China2University of Chinese Academy of Sciences, Beijing 100049, China3Beijing Jishuitan Hospital, The 4th Clinical Hospital of Peking University Health Science Center, Beijing 100035, China4School of Aerospace Engineering, Beijing Institute of Technology, Beijing 100081, China

Correspondence should be addressed to Yajun Liu; drliuyajun@163.com

Received 17 February 2019; Revised 22 May 2019; Accepted 24 June 2019; Published 14 August 2019

Guest Editor: Keiichiro Matoba

Copyright © 2019 ShunhaoWang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Diabetic foot ulcers (DFUs), the most serious complication of diabetes mellitus, can induce high morbidity, the need to amputatelower extremities, and even death. Although many adjunctive strategies have been applied for the treatment of DFUs, the lowtreatment efficiency, potential side effects, and high cost are still huge challenges. Recently, nanomaterial-based drug deliverysystems (NDDSs) have achieved targeted drug delivery and controlled drug release, offering great promises in varioustherapeutics for diverse disorders. Additionally, the radial extracorporeal shock wave (rESW) has been shown to function as arobust trigger source for the NDDS to release its contents, as the rESW harbors a potent capability in generating pressure wavesand in creating the cavitation effect. Here, we explored the performance of oxygen-loaded nanoperfluorocarbon (Nano-PFC)combined with the rESW as a treatment for DFUs. Prior to in vivo assessment, we first demonstrated the high oxygen affinityin vitro and great biocompatibility of Nano-PFC. Moreover, the rESW-responsive oxygen release behavior from oxygen-saturated Nano-PFC was also successfully verified in vitro and in vivo. Importantly, the wound healing of DFUs wassignificantly accelerated due to improved blood microcirculation, which was a result of rESW therapy (rESWT), and thetargeted release of oxygen into the wound from oxygen-loaded Nano-PFC, which was triggered by the rESW. Collectively, theoxygen-saturated Nano-PFC and rESW provide a completely new approach to treat DFUs, and this study highlights theadvantages of combining nanotechnology with rESW in therapeutics.

1. Introduction

Diabetic foot ulcers (DFUs), the most common complicationof diabetes mellitus, are caused by peripheral neuropathy,small vessel occlusion, and secondary infection or trauma,and they may lead to lower extremity amputation [1–4].Many adjunctive strategies have been developed for the treat-ment of DFUs in the clinical practice, including negativepressure wound therapy, ultrasound, recombinant humanplatelet-derived growth factor-BB, and acellular matrix

products [5–7]. However, low efficiency, potential sideeffects, and high cost have limited their wide application.Hypoxia is a key inhibiting factor for wound healing ofDFUs, which can block fibroblast proliferation, collagen pro-duction, and capillary angiogenesis and enhance the risk ofinfection [8–10]. Hyperbaric oxygen therapy (HBOT) is themost commonly utilized adjunctive therapy for improvingwound tissue hypoxia in DFU treatment [11–13], but thismethod has not achieved universal success in many studiesand is costly [14, 15]. And the production of HBO-related

HindawiOxidative Medicine and Cellular LongevityVolume 2019, Article ID 5738368, 10 pageshttps://doi.org/10.1155/2019/5738368

oxidative stress is a concern [16]. Thus, to improve treatmentefficiency and reduce costs, a need exists for a new and effec-tive DFU treatment method.

A nanomaterial-based drug delivery system (NDDS)could improve DFU treatment by means of targeted drugdelivery and controlled drug release [17–19]. Recently, grow-ing attention in the NDDS has given rise to the study ofmicro/nanobubbles, especially oxygen-filled nanobubbles[8, 20–22]. Among them, nanoperfluorocarbon (Nano-PFC)has been extensively explored as an oxygen-loaded system toovercome hypoxia-associated resistance in cancer therapiesowing to its high oxygen affinity and great biocompatibility[22–24]. Additionally, oxypherol (Fluosol-43), a type ofPFC, has obtained US Food and Drug Administration(FDA) approval for improving myocardial oxygenation andpreventing abnormalities in ventricular function [25]. Thus,Nano-PFC can be used as anNDDS to delivermolecules, suchas drugs and oxygen, to target tissues and release the contentsin response to physical stimuli [20, 23, 26].

The radial extracorporeal shock wave therapy (rESWT)has been widely applied in musculoskeletal disorders, myo-cardial infarction, wound healing, and erectile dysfunctiondue to the noninvasive mode of treatment, cost effectiveness,lower energy level, and negligible side effects [27–29]. Moreimportantly, rESW is a type of pneumatically generated pres-sure wave [30, 31] that can induce the cavitation effect, whichrefers to the rapid formation, expansion, and collapse ofsmall vapor bubbles in liquids due to sharp pressure changes[32–34]. Thus, the rESW is a candidate trigger source toenhance inclusion release inside an NDDS. Furthermore,ESW therapy has been reported to distinctly improve andpromote the healing of DFUs by mainly enhancing bloodmicrocirculation and tissue regeneration and reducing oxida-tive stress [35, 36]. However, the corresponding studies aboutrESWT are few.

Herein, we developed a completely new strategy for thetreatment of DFUs by combining the rESW with oxygen-loaded Nano-PFC, which could effectively improve bloodflow and provide a targeted supply of oxygen. In our study,the rESW-responsive oxygen release feature of Nano-PFCwas first proved in vitro and in vivo. Moreover, rESWT wasdemonstrated to improve blood microcirculation and accel-erate the wound healing of DFUs. Based on our results, therESW-responsive oxygen-loaded Nano-PFC can provide anew sight and great potential for DFU treatment.

2. Materials and Methods

2.1. Preparation and Characterization of Nano-PFC. PFC(300 μL perfluoro-15-crown-5-ether, Fluorochem, UK) wasadded to 1% human serum albumin (HSA) (Sigma-Aldrich,China) in 0.01 M phosphate-buffered saline (PBS) solution(4mL). Then, the mixture was emulsified by an ultrasonichomogenizer (Scientz-1200E, China) for 200 s after slightoscillation. The obtained emulsion was centrifuged(8000 r/min) for 3min, and then, the precipitate was resus-pended in PBS for further use. The size and morphology ofNano-PFC were characterized by transmission electronmicroscopy (TEM) (SU-8020, Hitachi, Japan) after negative

staining using 1.5% phosphotungstic acid and Malvernzetasizer (NANO ZS, UK).

2.2. Measurement of Oxygen Release from Nano-PFCTriggered by rESW In Vitro. Nano-PFC solution (4mL, con-taining 300 μL of PFC) was stored in an aseptic oxygenchamber (O2 flow rate = 5 L/min) for 5min for Nano-PFCoxygenation. The oxygen-free water was obtained afterN2 bubble. Then, oxygen-saturated Nano-PFC was addedto 45mL of oxygen-free water. The oxygen concentrationwas measured by a portable dissolved oxygen meter(Rex, JPBJ-608, China) before and after adding Nano-PFCwith or without rESW (MASTERPULS® MP100, STORZMEDICAL AG, Switzerland) treatment (1 bar, 2Hz, 25min).

2.3. Cellular Experiments. Murine breast cancer (4T1) andhuman umbilical vein endothelial (HUVE) cells were pur-chased from American Type Culture Collection (ATCC)and cultured with 1640 medium containing 10% fetal bovineserum (FBS, HyClone) at 37°C in a 5% CO2 atmosphere. The4T1 and HUVE cells were seeded into separate 96-well plates(0 8 × 104 cells/well). After 24 h, the different amounts ofNano-PFC (0.25, 0.5, 1, 2, and 4 μL) were added to the96-well plates, and the cells were incubated for 24 h(n = 4). The cell viability was determined by the WST-8(4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) assay fol-lowing a standard protocol (Solarbio, 1000T, China).

2.4. Animal Model. All of the animal experiments wereapproved by the Animal Ethics Committee of the ResearchCenter for Eco-Environmental Sciences, Chinese Academyof Sciences. Sixteen female albino Wistar rats (180–220 g)were purchased from the Peking University LaboratoryAnimal Research Center and randomly divided into Ctrl,rESW, Nano-PFC@O2, and rESW+Nano-PFC@O2 groups(n = 8). The rats in all groups were intraperitoneally injectedwith streptozotocin (STZ) solution (Sigma-Aldrich, 60mg/kgbody weight) that was freshly dissolved in 0.1mg/L citrate-sodium citrate buffer (pH 4.5) for approximately 2 months.With respect to the blood glucose level, measured by aglucometer, it was ≥300mg/dL for two consecutive weeks,suggesting that the STZ-induced diabetic rat model was suc-cessfully established, in agreement with previous reports[10]. In order to faithfully reflect the realistic complex pathol-ogies and mimic those conditions under diabetes mellitus, wedid not actually add additional treatments. Therefore, duringthis period, we kept monitoring blood glucose levels withoutadditional work. Nonetheless, the protocol of establishing aSTZ-induced diabetic rat model has been widely applied inthe wound healing studies of diabetic foot ulcers, assupported by the following references (Table S1) [10–17].Then, a wound (5 × 5mm) was created in the left forepawof each rat after anesthetizing with sodium pentobarbital(45mg/kg body weight) by intraperitoneal injection. Thesize of the wound was measured by a Vernier caliper.BALB/c female mice (7-8 weeks old) were purchasedfrom the Vital River Laboratory Animal Technology Co.Ltd. (Beijing, China). The 4T1 tumor-bearing mouse model

2 Oxidative Medicine and Cellular Longevity

was established following the instructions in our previousreports [37].

2.5. In Vivo Photoacoustic (PA) Imaging. When the tumorvolume of 4T1 tumor-bearing mice reached up to 100mm3,the tumor oxygenation levels were detected using a PAimaging system (Vevo 2100, FUJIFILM VisualSonics Inc.,Canada) utilizing the oxy-hemo mode (750 and 850nm).The 4T1 tumor-bearing mice were injected with oxygen-saturated Nano-PFC through the tail vein (200 μL, contain-ing 30μL of PFC), and the mice breathed pure oxygen for20min before PA imaging.

2.6. Blood Flow by Laser Doppler Imaging. The blood flow ofthe wound was measured by laser Doppler imaging (MoorLDI V3.01, UK). STZ-induced diabetic rats were injectedwith oxygen-saturated Nano-PFC through the tail vein(500 μL, containing 80μL of PFC), and the mice breathedpure oxygen for 20min before imaging. The rats in therESW+Nano-PFC@O2 group were treated with rESW for20min (1 bar, 2Hz). The quantitative data were obtainedfrom Moor FLPI measurement software (Version 2.1).

2.7. Immunohistochemistry Analysis. After various treat-ments for 4T1 tumor-bearing mice, the mice were sacrificedand the tumors were isolated from the mice on the 15thday and then fixed in 4% paraformaldehyde, embedded,and sectioned at 8 μm. Next, the immunohistochemical anal-ysis of HIF-1α for tumor tissues was performed as the proto-col. The expression levels of HIF-1α were quantified byImageJ software (https://imagej.nih.gov/ij/).

2.8. Nano-PFC plus rESW Treatment In Vivo. The STZ-induced DFU rats were anesthetized with sodium pentobar-bital (45mg/kg body weight) by intraperitoneal injectionprior to treatment. Thereafter, the rats were injected withPBS (500 μL) through the tail vein for the control group.The rESW group received rESW treatment (1 bar, 2Hz,20min). The rats were injected with oxygen-saturatedNano-PFC through tail vein intravenous injection (500 μL,containing 80 μL of PFC) and subjected to pure oxygenbreath for 20min in the Nano-PFC@O2 group. The rESW+Nano-PFC@O2 group denoted the rats that inhaled pureoxygen for 20min after Nano-PFC@O2 injection (500 μL,containing 80μL of PFC). Afterwards, the rats were treatedwith rESW (1 bar, 2Hz) for 20min. All administration andtreatments were conducted once every other day for a total

of three times. The size of the wound was measured by aVernier caliper.

2.9. Statistical Analysis. The statistical analysis of experimen-tal data was determined by an independent t-test or one-wayANOVA test using the SPSS Statistics 17.0 software. All ofthe data are presented as the mean ± standard error. Statisti-cal significance was determined with P < 0 05 and P < 0 001.

3. Results and Discussion

3.1. Synthesis and Characterization of Nano-PFC. The nano-droplets of HSA-stabilized Nano-PFC were obtained usingthe modified microemulsion method under ultrasonication,as described in previous reports (Figure 1) [22, 23].Figures 2(a) and 2(b) show that the synthesized HSA-stabilized Nano-PFC exhibits a highly uniform size distri-bution with an average diameter of about 70 nm, as measuredby TEM and dynamic light scattering (DLS). Moreover, thesize of Nano-PFC changed negligibly after rESW treatment,which proved thatNano-PFChad great stability (Figure 2(b)).

3.2. rESW-Responsive Oxygen Release of Nano-PFC In Vitroand In Vivo. PFC, as an artificial oxygen carrier, has beenused in the clinical treatment of ischemic diseases such ashemorrhagic shock and allogeneic blood transfusions owingto its excellent dissolving capacity for many gases, includingoxygen and carbon dioxide [25, 38]. Thus, the abilities ofNano-PFC for oxygen loading and rESW-responsive oxygenrelease (Figures 1 and 2(c)) were measured by an oxygenmeter. The concentration of pure oxygen saturation withinNano-PFC was about 1.50mg/mL at 25°C (1 atm), whichwas consistent with previously reported results [23]. Asshown in Figure 2(c), the dissolved oxygen concentration inwater speedily increased in a short time, and then, the oxygenwas slowly released from the oxygen-loaded nanodropletsover time when the oxygen-loaded Nano-PFC was added tooxygen-free water. Importantly, burst-like oxygen releasein a dose-dependent manner was observed under rESWtreatment with different frequencies (2, 4, 6, 8, and 10Hz).These results demonstrated the high oxygen solubility andrESW-responsive oxygen release abilities of Nano-PFC.Moreover, after one injection, Nano-PFC may reload oxygenwithin the lung capillaries during blood circulation. Thus,oxygen-filled Nano-PFC can deliver oxygen to the targetedarea and then reversibly release oxygen triggered by therESW. Compared to HBOT, this Nano-PFC-based treatment

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3Oxidative Medicine and Cellular Longevity

strategy can effectively reduce the cost of treatment and avoidserious side effects, such as barotrauma, central nervoussystem and pulmonary oxygen toxicity, and increased riskof claustrophobia [39]. In addition, the cell viability of 4T1and HUVE cells was not significantly affected by the dif-ferent doses of Nano-PFC, which exhibited that Nano-PFC has great biocompatibility for different types of cells(Figure 2(d)).

Inspired by the above great performance in vitro, we fur-ther researched the oxygen release behavior in vivo based ona 4T1 tumor-bearing mouse model. It is worth mentioningthat nanosized materials can achieve highly efficient accumu-lation in tumor tissue depending on the enhanced permeabil-ity and retention (EPR) effect [40]. Additionally, almost all ofthe solid tumors have a hypoxic region due to the deficientblood supply along with the tumor progression [41, 42].

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4 Oxidative Medicine and Cellular Longevity

Likewise, the disturbance of the vascular system is one of thedirect reasons for DFUs because it can cause sustained oxy-gen deficiency and finally chronic hypoxia in the wound area[43]. However, we had established the wound-healingmodels of DFUs in the rats’ forepaws, which were too largeto be placed in the detector of the photoacoustic (PA)

imaging system. And the scientific rationale is very sound,for the microenvironment, the microstructure, and thepathology are very similar between tumors and diabeticfoot ulcers, such as hypoxia, angiogenesis, and inflammation[44–46]. Here, a 4T1 tumor-bearing mouse model was estab-lished by subcutaneously injecting 4T1 tumor cells, which

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Figure 3: Determination of oxygen release from nanovehicles with the aid of rESW. (a) Schematic illustration of rESW-respective oxygenrelease from oxygen-saturated Nano-PFC in 4T1 tumor-bearing mice. (b) PA imaging of 4T1 tumors for determining tumor oxygenationstatus by measuring the ratios of oxygenated hemoglobin (λ = 850 nm) and deoxygenated hemoglobin (λ = 750 nm) before and after rESWtreatments (20min). Scale bars are 3mm. The expression levels of HIF-1α in the tumors of mice from various groups were analyzed bythe immunohistochemical method. Dark blue arrowheads point at the HIF-1α-positive cells. Scale bars are 100 μm. (c) Quantification ofHIF-1α-positive cells in immunohistochemical images by ImageJ software.

5Oxidative Medicine and Cellular Longevity

belonged to the superficial tumor. And we have proved thatrESW could effectively improve local blood flow, which couldmake more oxygen-loaded Nano-PFC deliveries to thewound location and greater oxygen release under rESWtreatment. In support of this finding, our latest study uncov-ered that rESW could promote vessel vasodilation, tumorblood supply, and nanoparticle extravasation into tumormicroenvironment [47]. Therefore, we selected the 4T1tumor-bearingmousemodel as an alternativemethod to eval-uate the in vivo behavior of oxygen release and improvementof the hypoxia level as a result of rESW-responsive Nano-PFC. In this process, Nano-PFC can be used as a nanosizedcarrier system to deliver oxygen into tumor tissue andimprove tumor oxygenation under the assistance of thelocally applied rESW (Figure 3(a)). The nanodroplets canreversibly release oxygen within the tumor tissue, triggeredby the rESW, when Nano-PFC is injected into blood circula-tion. Thus, the 4T1 tumor-bearing mice breathing pure

oxygen were injected with 200 μL Nano-PFC (containing30μL of PFC) through the tail vein. Then, the tumor oxygen-ation status of each mouse was detected by PA imaging afterthe tumor region was treated with the rESW (1 bar, 4Hz)for 20min. PA imaging is noninvasive and is suitable forevaluating the blood oxygenation status by capturing theabsorbance of hemoglobin at 750nm (deoxygenated sta-tus) and 850 nm (oxygenated state) [23]. As shown inFigure 3(b), the oxygenation levels over the whole tumorregion rapidly increased in the group with hyperoxic breath-ing plus tumor-localized rESW treatment (rESW+Nano-PFC@O2) compared with the group with only hyperoxicbreathing (Nano-PFC@O2). We also evaluated the expres-sion level of hypoxia-inducible factor 1-alpha (HIF-1α), amarker of tumor hypoxia status, by analyzing the immuno-histochemistry after 15 days of treatment. In Figures 3(b)and 3(c), the expression levels of HIF-1α dropped 58.3% inthe rESW+Nano-PFC@O2 group in comparison with the

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6 Oxidative Medicine and Cellular Longevity

Nano-PFC@O2 group (P < 0 001). These results uncoveredthe fact that the rESW could effectively stimulate oxygenrelease from oxygen-loaded Nano-PFC and enhance tumoroxygenation status in vivo, which means that theoxygen-loaded Nano-PFC plus rESW could improve thehypoxic microenvironment around DFUs and accelerate thewound healing.

3.3. Blood Microcirculation by Laser Doppler Imaging.Furthermore, it has been proven that the rESW could inducetissue regeneration and improve blood microcirculation[28, 47–49]. Thus, laser Doppler imaging was used tomeasurethe change in blood flow after different treatments. Thedata revealed that the blood microcirculation of the rat’sforepaw was improved about 5.4 times after rESW treatmentcompared to the Nano-PFC@O2 group without the rESW(Figure 4, P < 0 001). Nearly every step in the woundhealing process requires oxygen for inducing angiogenesisby increasing vascular endothelial growth factor (VEGF)expression [9, 50]. Thus, rESW-responsive improvement ofblood microcirculation and oxygen reversible release fromNano-PFC provided the potential for accelerating footwound healing in STZ-induced DFU models.

3.4. Nano-PFC plus rESW Treatment Accelerated DiabeticFoot Wound Healing. Nano-PFC@O2 and rESW are the keyfactors in our strategy for the treatment of DFUs. Thus, wefurther evaluated whether Nano-PFC plus rESW treatmentcould bring any advantages for the wound healing of DFUs.First, the rat models of STZ-induced DFU were established,and then, a wound was created in each rat’s forepaw. Asshown in Figure 5, the intervening treatments includingrESW, Nano-PFC@O2, and rESW plus Nano-PFC@O2 inor-dinately accelerated the wound healing of DFUs, comparedto diseased rats with DFUs that did not receive any treat-ment, namely, the untreated control (Ctrl). Moreover, thewound healing effect of rESW and PFC was also consistentwith previous studies [2, 43, 51–57]. And the rate of woundhealing in the rESW+Nano-PFC@O2 group was significantlyhigher than that in the Nano-PFC@O2 group and rESWgroup (Figure 5; #P < 0 001 and ∗P < 0 05). The improve-ment of wound healing might be attributed to the rESWand the oxygen supply from Nano-PFC. First, rESW treat-ment could increase blood microcirculation in the woundhealing process. Meanwhile, the oxygen supply from Nano-PFC could neutralize the hypoxic microenvironment due tothe vascular disruption and the oxygen consumption by

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7Oxidative Medicine and Cellular Longevity

inflammatory and stromal cells within the wound. AndrESW treatment might reduce oxidative stress, compared toHBOT, which overcame the drawbacks of HBOT [35, 36].

At present, although many adjunctive therapies havebeen applied for the healing of DFUs, just as previously dis-cussed [5–7, 12], the clinic treatment of DFUs remained asan enormous challenge due to the multifactors of that. Fortu-nately, recent studies found that ESW therapy was an effec-tive treatment approach for DFUs, being more effectivethan HBOT in clinical observations [2, 58, 59]. Moreover,focused ESW was used as a major therapeutic modality inmost studies. Thus, herein, we garnered a new sight intothe treatment of DFUs. On the other hand, PFC-based bioa-gents have been shown to be effective in the treatment ofchronic wound [43, 51, 60]. Importantly, PFC has beenclinically approved by FDA [23, 61]. Based on the abovedesirable features, we therefore developed the rESW forDFU treatment in combining oxygen-saturated Nano-PFC,which brought a completely new treatment strategy forDFUs. To this end, this strategy provided a great potentialfor further clinical trials. However, the potential safety issuesof PFC-based micro/nanomaterials should not be neglected.Thus, we will continue this study forward.

4. Conclusions

In conclusion, the Nano-PFC developed herein exhibiteduniform size and achieved the abilities of high-efficiency oxy-gen loading and rESW-responsive oxygen reversible releasein vitro and in vivo. More importantly, the synergistic effectof the rESW and Nano-PFC accelerated foot wound healingin STZ-induced type 1 diabetes mellitus rats by improvingblood microcirculation and providing a targeted supply ofoxygen to the wound. This study proved the benefits ofcombining rESW and Nano-PFC to treat DFUs, which is anovel strategy for the treatment of diabetic foot ulcers.

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request.

Conflicts of Interest

The authors report no conflict of interests.

Authors’ Contributions

Shunhao Wang and Chunyang Yin contributed equally tothis work.

Acknowledgments

The authors thank Prof. Zhuang Liu and Dr. Xuejiao Song(Soochow University) for the help in the synthesis ofNano-PFC. And we thank LetPub (http://www.letpub.com)for its linguistic assistance during the preparation of thismanuscript. This research was supported by grants fromthe National Natural Science Foundation of China (GrantNos. 21425731, 21637004, 21621064, and 11772343), the

Strategic Priority Research Program of the Chinese Academyof Sciences (Grant No. XDB14000000), and the BeijingMunicipal Science and Technology Commission ResearchFoundation (Z181100006218014 and Z171100000417025).

Supplementary Materials

Supplementary description. Table S1: references relatedto the wound healing studies of diabetic foot ulcers.(Supplementary Materials)

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